Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of airway inflammation of asthma. The long-term goal of this research is to understand the regulation of iNOS activity and to devise novel methods to regulate it. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its cellular regulation. We have recently shown that iNOS is degraded through the ubiquitin-proteasome pathway. The specificity of the ubiqutination system, is mainly provided by the specific ubiquitin ligase enzyme (E3) that recognizes and binds to the target protein. Therefore, identification and characterization of the E3 ubiquitin ligase for iNOS should be regarded as one of the most critical steps for understanding the mechanisms of iNOS cellular regulation. Our preliminary data identified an F-box-containing protein;we termed FBXinos that is a likely candidate to be the E3 ubiquitin ligase for iNOS. Additional preliminary data suggest that cells regulate NO synthesis by temporal and spatial regulation of iNOS. These mechanisms include a relatively rapid rate of iNOS turnover and sequestration of iNOS to a perinuclear location we termed the """"""""physiologic aggresome"""""""". Interestingly, our preliminary data suggest that FBXinos is important not only for iNOS turnover but also for subcellular targeting of iNOS. We propose to test the following hypothesis: An F-box-containing protein, FBXinos, is the E3 ligase for iNOS and it plays a central role in iNOS cellular regulation. The cellular regulation of this FBXinos further modulates iNOS and NO cellular levels. To test this hypothesis we propose studies with the following specific aims: 1) Characterization of the regulation of iNOS by FBXinos. 2) Determine the mechanisms by which FBXinos ubiquitin ligase targets iNOS to the aggresome. 3) Characterization of the regulation of iNOS and FBXinos in airway inflammation. Studies will be conducted in cultured cells expressing iNOS and in primary bronchial epithelial cells cultured at the air/liquid interphase. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handling of iNOS. Future therapeutic strategies can be designed to regulate these cellular responses in disease states.
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